Resting B cells must pass two G1 checkpoints prior to initiating DNA duplication. Checkpoint 1 (the G1 restriction point) requires a minimal level of active D-type cyclin/cdk4/6 kinase complexes while checkpoint 2 (G1/S boundary) requires activated cyclin E/cdk2 kinase complexes. In the first specific aim, resting hen egg lysozyme (HEL) specific, transgenic B cells (HEL B cells) will be used to test the hypothesis that soluble antigen initiates passage of resting B cells through checkpoint 1. The ability of soluble antigen to induce cell cycle related protein expression will be monitored by Western and IP-Western assays, the appearance of cyclin dependent kinase activities determined using in vitro kinase assays, and the composition of E2F complexes. Next, we will test the hypothesis that CD40-derived signals are required for B cells to pass checkpoint 2 and that this occurs in a p21 dependent fashion. Transgenic mice expressing mutated Cd40 fusion proteins will be used to characterize the signal transduction mechanism responsible for the CD40-mediated induction of p21. CD40-mediated signals likewise regulate the continued proliferation of cycling B cells, and in contrast to resting B cells, this occurs independently of D-type cyclin/cdk4/6 kinases. Experiments are proposed to determine the molecular mechanism(s) responsible for the selective induction of cdk2 by CD40 signaling of cycling B cells. For the second aim, We have acquired seven B cell lymphomas whose response to CD40-derived signals can be divided into three subgroups. Subgroup 1 B cell lymphomas require Th cell-derived signals to proliferate in vitro and grow in vivo. Subgroup 2 B cell lymphomas initially proliferate to CD40 stimulation, but undergo cell cycle arrest and/or apoptosis within 2 to 3 days. Subgroup 3 B cell lymphomas immediately undergo cell cycle arrest and/or apoptosis when stimulated through CD40. Experiments are proposed to characterize the effect of CD40-derived signals upon the cell cycle machinery of these three subgroups of B cell lymphomas. Thus, do differences in CD-40-mediated changes in the G1 cell cycle machinery account for the differential effects of CD40 signaling to these three subgroups of B cell lymphomas? The final aim will use hybrid mice expressing a ganciclovir-inducible suicide gene in all activated T cells to study the in vivo growth patterns of subgroup 1 and 2 B cell lymphomas in the presence and absence of T cells. It is hoped that this model system will enable us to study the linearity of the transformation process that is responsible for malignant B cell growth becoming T cell independent.